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Abstract

As one of the most serious types of primary bone tumor, osteosarcoma (OSA) features metastatic lesions, and resistance to chemotherapy is common. The underlying mechanisms of these characteristics may account for the failure of treatments and the poor prognosis of patients with OSA. It has been reported that inhibition of Cyr61 suppresses OSA cell proliferation as it represents a target of statins. In addition to cystein‑rich protein 61 (Cyr61) and nephroblastoma overexpression, connective tissue growth factor (CTGF) is a member of the CCN family and may therefore exhibit effects on human OSA cells similar to those of Cyr61. In the current study, acridine orange/ethidium bromide staining were used to determine the rate of apoptosis. The present study demonstrated that small interfering RNA‑mediated silencing of CTGF promoted cell death and suppressed OSA cell migration and invasion, as indicated by wound healing and Transwell assays, while lentivirus‑mediated overexpression of CTGF reversed these effects. Furthermore, a colorimetric caspase assay demonstrated that CTGF knockdown enhanced the efficacy of chemotherapeutic drugs. The results of the present study provided a novel molecular target which may be utilized for the treatment of metastatic OSA.

Introduction

Osteosarcoma (OSA) is globally one of the most
common types of primary bone tumor and is predominantly observed in
children and adolescents (<20 years old) (1). Patients with localized disease have a
five-year recurrence-free survival rate of 80%; however, the
prognosis of OSA is poor in metastatic osteosarcoma. In spite of
OSA occurring in any type of bone in the body, the metaphyseal
(actively growing) regions of the distal femur, proximal tibia and
proximal humerus are the most frequent origins of the primary tumor
and the sites with the highest probability of metastasis are the
lungs and distant bones (2).

It has been reported that several genes are able to
regulate cell proliferation and differentiation; these genes carry
numerous mutations associated with significant neoplasmic
abnormalities in OSA (3–9). Of note, mutations in tumor suppressor
genes, including p53, MDM2 and riboblastoma protein have been
reported to have major roles in the tumorigenesis of OSA (3,4). OSA
is also associated with the aberrant expression of certain
transcription factors expressed in bones, including c-fos, whose
overexpression has been shown to result in OSA in the bones of mice
(5), as well as osteoblast
differentiation factor osterix (6,7). In
OSA cell lines, Runx2 was found to be downregulated or
dysfunctional (8), and in
high-grade pediatric OSA, genomic aberrations in the Twist have
been reported (9).

Resistance to conventional chemotherapy is one of
the characteristics of metastatic OSA and represents a considerable
obstacle for its clinical treatment (10). However, only a small number of
genes, including HES1 (11–13)
and Ezrin (10) have been
implicated in the progression and metastasis of OSA.

It has been reported that statins exert anti-tumoral
effects on OSA cells (13–15). Cystein-rich protein 61 (Cyr61), a
member of the Cyr61/connective tissue growth factor
(CTGF)/nephroblastoma overexpressed (NOV) (CCN) family of secreted
proteins, was among the factors downregulated by statins. This CCN
protein family comprises Cyr61, CTGF, NOV and Wnt-induced secreted
proteins (WISP)1, -2 and -3 (16).
As a member of the CCN family, CTGF was hypothesized have effects
on osteocarcinoma similar to those of statins. The present study
therefore assessed the effects of CTGF knockdown or
lentivirus-mediated overexpression of CTGF as well as statin
treatment on the biological properties of OSA cells.

TRIzol reagent (Invitrogen; Thermo Fisher
Scientific) was used to isolate RNA according to the manufacturer's
instructions, which was stored at −20°C. cDNA was synthesized using
3 µg RNA, which was denaturated and reverse-transcribed by
using 300 U Moloney murine leukemia virus reverse transcriptase, 15
mg oligo dT primers and 1 mM deoxynucleoside triphosphate (dNTP)
(Promega, Madison, WI, USA) in a total volume of 30 µl. SYBR
Green Master Mix kit (ABGen, Courtaboeuf, France) was used for
qPCR. A total of 0.5 mM of each primer (Invitrogen; Thermo Fisher
Scientific) was used with sequences as follows: Human CTGF, forward
5′-CAG GCT AGA GAA GCA GAG CC-3′ and reverse 5′-GTA ATG GCA GGC ACA
GGT CT-3′; β-actin, forward 5′-CTC CAT CCT GGC CTC GCT GT-3′ and
reverse 5′-GCT GTC ACC TTC ACC GTT CC-3′. Thermocycling was
conducted using the ABI 7500 (Applied Biosystems; Thermo Fisher
Scientific) and the cycling conditions were as follows: Initial
denaturation at 95°C for 15 min, followed by 40 cycles of 20 sec at
95°C, 15 sec at 58°C and 15 sec at 72°C, and final extension at
72°C for 7 min. The 2−ΔΔCt method was used to determine
the relative quantities of RNA.

Plasmid transduction

In order to investigate the role of CTGF in OSA,
cell lines were transduced with lentiviral vectors (LV) encoding
either the full-length sequence (LV-CTGF) or a specific short
hairpin (sh)RNA (sh-CTGF). The full-length CTGF ORF (1047 base
pairs; GenBank accession number, CR541759.1) was amplified from the
pFLAG-CMV2-CTGF plasmid (Invitrogen; Thermo Fisher Scientific). The
primer sequences were as follows: Forward,
5′-TACTGGCGGCGGTATACCCG-3′ and reverse, 5′-TGCCATGTCTCCGTACAT-3′.
The PCR product was inserted into the expression vector
pcDNA3.1/myc-His(-)B-3X FLAG-IRES-hrGFP, derived from
pcDNATM3.1/myc-His(-)B (Invitrogen; Thermo Fisher Scientific). Cell
transduction was performed using Lipofectamine 2000 (Invitrogen;
Thermo Fisher Scientific) according to the manufacturer's
instructions.

Proliferation assay

A bromodeoxyuridine (BrdU) incorporation assay was
used to quantify cell replication. A previously described procedure
was used in the present study (17). In brief, cells were cultured for 24
h in the presence of increasing concentrations of bisphosphonates
(10−9–10−4 M) and labeled with BrdU for the
last 6 h (kit purchased from GE Healthcare Life Sciences,
Roosendaal, The Netherlands).

Detection of apoptosis and necrosis

Double staining with ethidium bromide and acridine
orange was performed to visualize and quantify the number of viable
cells (green nuclei), apoptotic cells (nuclei condensed and colored
orange), and necrotic cells (red nuclei). In briefly, 2 µl
dye mixture (100 µg/ml acridine orange and 100 µg/ml
ethidium bromide) was added to 20 µl cell suspension and
immediately examined with the 40X oil immersion objective using a
Leitz DMRB fluorescence microscope (green/red filter; 100 W lamp;
Leica Microsystems GmbH, Wetzlar, Germany) equipped with a
photometrics CCD camera and the Logikon image analysis system
(Numeris Benelux SA, Ath, Belgium). Several fields, randomly
chosen, were digitized and 600–800 nuclei for each sample were
counted and scored. Results were expressed as the relative
percentages of viable, apoptotic and necrotic cells to the total
number of cells scored.

Caspase activity

Effector caspase activity was performed as
previously described (14,15). In brief, cells were treated with 10
mM atorvastatin (Adooq BioScience LLC, Irvine, CA, USA) or the
solvent for 24 h then the caspase activity was determined. Cellular
extracts (50 µg) were incubated with 0.2 mM
acetyl-Asp-Glu-Val-Asp-p-nitroanilide (caspases-3, -6 and -7; Enzo
Life Sciences, Inc., Farmingdale, NY, USA), Ac-LEHD-pNA (caspase-9;
Enzo Life Sciences, Inc.) or Ac-IETD-pNA (caspase-8; Enzo Life
Sciences, Inc.) as the substrates for the previously reported times
(14,15) at 37°C in the presence or the
absence of the specific caspase inhibitors Ac-DEVD-CHO, Ac-LEHD-CHO
and Ac-IETD-CHO (10 µM). The specific activity (nmol of
pNA/min/mg protein) was expressed as treated over control
ratios.

Migration and invasion assays

A wound-healing assay was performed following the
manufacturer's instructions (ibidi GmbH, Martinsried, Germany). A
Transwell migration and invasion assay as performed as described
previously (14). In brief, the
cells (50,000 cells/insert) were incubated 2 h with or without
statin and/or z-VAD-fmk prior to seeding into the inserts and
incubation for a further 22 h. The cells that did not migrate
through the filter were removed from the upper surface of the
membrane using cotton-tipped swabs. The cells migrated to the
underside were fixed in 3.7% paraformaldehyde in phosphate-buffered
saline (PBS) at 4°C and stained with crystal violet (Amresco,
Solon, OH, USA). The membranes were then cut from the insert and
observed under a microscope (Axioplan 2 Imaging Mot Microscope
System; Zeiss, Oberkochen, Germany). Five fields were randomly
selected and counted and each assay was performed in duplicate.

Statistical analysis

Values are expressed as the mean ± standard
deviation. Two-factor analysis of variance was used to compare
values between groups, using SPSS software, version 19.0 (IBM SPSS,
Armonk, NY, USA). P<0.05 was considered to indicate a
statistically significant difference between values.

Results

CTGF expression is reduced by
atorvastatin (statin) in OSA cells

RT-qPCR analysis of CTGF was performed in the SaOS2,
U2OS, CAL72, MG63 and OHS4 human OSA cell lines, revealing that
CTGF mRNA was expressed in all cell lines, particularly in SaOS2
cells (Fig. 1A). Furthermore, the
effect of statin treatment on the expression of CTGF was assessed
in the OSA cell lines. CTGF mRNA expression in the panel of OSA
cell lines was markedly decreased following treatment with statin
(10 mM) (P<0.05 vs. untreated) (Fig. 1B). In addition, the effect of
statin (10 mM) on the protein levels of CTGF in the panel of cell
lines was assessed by immunoblot analysis, revealing that the
protein levels of CTGF were decreased following statin (Fig. 1C). Collectively, these results
indicated that statin treatment led to the downregulation of CTGF
in human OSA cells. As the SaOS2 and U2OS cell lines expressed the
highest and lowest levels of CTGF, respectively, they were selected
to be used in the subsequent experiments.

CTGF expression does not affect OSA cell
proliferation

A BrdU incorporation assay were used to determine
the proliferative rates of transduced and parental cells, revealing
that these were not affected by plasmid transduction (Fig. 2A). The results therefore indicated
that CTGF had no significant effects on OSA-cell proliferation in
human cell lines.

Evasion of apoptosis by OSA cells is
dependent on CTGF expression

The present study investigated the effects of CTGF
on OSA cell death. As shown in Fig.
2B, apoptotic and necrotic indices of sh-CTGF-transduced cells
were higher than those of parental cells. By contrast,
LV-CTGF-transduced cells displayed lower apoptotic and necrotic
indices compared with those of parental cells. Furthermore, it was
revealed that sh-CTGF-transduced cells exhibited increased caspase
activity and an elevated Bax/Bcl2 ratio compared with those of
parental cells. By contrast, caspase activity and the Bax/Bcl2
ratio were reduced in CTGF-overexpressing OSA cells compared with
those in parental cells (Fig. 2C and
D). These results indicated that CTGF expression was associated
with the evasion of apoptosis by OSA cells.

The dose-dependent cytotoxic effects of doxorubicin,
cisplatin and methotrexate on OSA cell viability are utilized for
the chemotherapeutic treatment of OSA (14). The present study revealed that CTGF
silencing significantly enhanced the caspase activity in SaOS2
cells following treatment with doxorubicin, cisplatin or
methotrexate, whereas LV-CTGF slighly decreased caspase levels
compared with those in native SaOS2 cells treated with the
chemotherapeutics (Fig. 3A–C). It
is therefore concluded that silencing of CTGF enhanced the efficacy
of chemotherapeutic drugs against OSA.

Cell migration and invasion are dependent
on CTGF expression in vitro

The present study further investigated the
invasiveness and migratory potential of transduced OSA cell lines,
which represent the main characteristics of OSA progression and the
development of metastasis. The results showed that CTGF silencing
inhibited wound healing in sh-CTGF-transduced cells compared with
that in parental cells, while CTGF overexpression enhanced wound
healing (Fig. 4A). In addition,
CTGF overexpression enhanced the migratory potential in a Transwell
assay (Fig. 4B). The observed
inhibition of the migratory potential by statin was not able to be
rescued by overexpression of CTGF (Fig. 4C). Furthermore, a Transwell assay
using Matrigel-coated inserts revealed that silencing of CTGF
inhibited the invasive capacity of OSA cells, while cell
invasiveness was promoted by CTGF overexpression (Fig. 4D). All of these results implied
that CTGF had positive effects on cell migration and invasiveness
in vitro, whereas invasion and migration were reduced in
CTGF-silenced OSA cells. It can be concluded that CTGF expression
is associated with the aggressiveness and metastatic potential of
OSA cells.

Discussion

Conserved cysteine residues covalently bound to
isoprenoids can be post-translationlly modified by prenylation,
which is essential for the pro-tumorigenic activity of certain
guanosine triphosphatases, including Ras and Rho-like proteins
(18,19). Synthetic bisphosphonates with
inhibitory activities on geranylgeranyltransferase type and
farnesyltransferase can be utilized as anti-cancer drugs which
partly block prenylation through inhibition of farnesyl
pyrophosphate (FPP) synthase activity; this approach is a novel
therapeutic strategy for several cancer types, including OSA and
bone metastases (20–25). Statins act as hypocholesterolemic
agents with inhibitory effects on the activity of
3-hydroxy-3-methylglutaryl-coenzyme A reductase (26) and represent another class of drug
which acts through depleting downstream isoprenoid residues,
including such as geranylgeranylpyrophosphate or FPP. Previous
studies on OSA reported that statins not only induced apoptosis but
also reduced cell migration and invasion, and potentiated the
effects of chemotherapeutic agents (13–15).
However, the anti-cancer efficacy of statins in vivo remains
to be clarified. Previous clinical studies indicated that statins,
apart from exhibiting anti-cancer effects, may also be associated
with an increased risk for the development of cancer de novo
(27–29). These conflicting results indicate
that the understanding of the mechanisms of action of statins is
required to be expanded and refined, and that novel targets for
cancer therapy require to be discovered.

Previous studies reported that Cyr61, which encodes
a secreted protein known to modulate tumor development and
progression, was downregulated by statins (30–32)
and that CTGF is also among the molecular targets of statins
(33,34). CTGF is a matricellular protein of
the CCN family of extracellular matrix-associated heparin-binding
proteins, which comprises Cyr61, CTGF, NOV and WISP1-3 (35–37).
CTGF has important roles in numerous biological processes,
including cell adhesion, migration, proliferation, angiogenesis,
skeletal development and tissue wound repair, and is critically
involved in fibrotic disease and several types of cancer (33,34,38).
Members of the CCN protein family have similar domains, indicating
that CTGF may have the similar roles in OSA cells to those of
Cyr61.

The present study enhanced or silenced the
expression of CTGF in human OSA cells to determine the role of CTGF
in OSA development and progression. A BrdU incorporation assay did
not reveal any significant effects of CTGF on the proliferation of
human OSA cell lines. By contrast, CTGF silencing slightly
increased OSA cell death and enhanced the anti-neoplasic and
pro-apoptotic effects of the chemotherapeutics doxorubicin,
cisplatin and methotrexate, which may represent a novel strategy to
enhance the efficacy of OSA treatments. A positive combinatory
effect of statins with chemotherapeutic drugs in OSA or other
cancer types has been indicated by previous studies (13,39–42).
The present study focused on CTGF expression in OSA cells,
independent of the presence of statins. As silencing of CTGF
enhanced the anti-tumoral effects chemotherapeutic drugs, it was
indicated that CTGF knockdown may reduce the resistance of OSA
cells to chemotherapy.

OSA bears the characteristics of rapid and frequent
development of metastatic lesions. In vitro experiments
performed in the present study demonstrated that the migratory and
invasive capacities of human OSA cells were reduced by CTGF
silencing, whereas CTGF overexpression led to an increase in cell
migration and invasion. By contrast, previous studies reported that
silencing or inhibition of CTGF reduced the motility and
invasiveness of breast and prostate cancer cells (43,44).
Due to this discrepancy, the roles of CCN family proteins,
particularly CTGF, in OSA require further study. In OSA cell lines,
Nov was reported to be expressed at variable levels (45) and may be associated with poor
prognosis and an increased risk of developing metastases (46). The predictive value of CTGF
expression levels with regard to the outcome and progression of
human OSA requires to be investigated in future studies analyzing
CTGF expression in primary and metastatic tumors.

In conclusion, the results of the present study
revealed that OSA cell invasion and migration was regulated by CTGF
in vitro. CTGF was indicated to have a critical role in the
genesis and progression of human OSA, and to be involved in the
evasion of apoptosis, aggressiveness and metastasis formation of
OSA. Targeting of CTGF may be a strategy to enhance the efficacy of
chemotherapeutics in the treatment of OSA as well as to reduce the
aggressiveness of OSA cells.